Techniques that target gene function at the level of DNA and mRNA provide powerful methods for modulating the expression of proteins encoded by specific genes. For example, the tet/dox and Cre/lox systems have been widely used to target gene expression at the transcriptional level (Ryding et al., 2001) and RNA interference is rapidly being adopted as a method to achieve post-transcriptional gene silencing (Fire et al., 1998; Madema, 2004).
However, methods for regulating protein function directly are limited, especially in mammalian cells. Inhibitors or activators of particular proteins have been identified, and often take the form of cell-permeable small molecules. Many of these molecules have found widespread use as biological probes, often because the speed, dosage-dependence, and reversibility of their activities, which complement methods for genetically modulating gene expression (Schreiber, 2003). However these inhibitors or activators are often promiscuous, affecting several proteins rather than a specific protein (Davies et al, 2000; Bain et al., 2003; Godl et al., 2003).
Shokat and coworkers have developed a method by which specific kinases can be inhibited using a small-molecule modulator (Shah et al., 1997; Bishop et al., 1998). This method involves mutating the protein of interest, typically replacing a large conserved residue in the active site with a smaller residue, such as glycine or alanine. Specificity is achieved by chemically modifying a promiscuous inhibitor to include a bulky side-chain substituent (e.g., R-group), which fills the corresponding cavity in the binding site of the modified protein of interest, while preventing productive interactions with other kinases. While this so-called “bump-hole” approach has been successful both in cultured cells and in mice (Bishop et al., 2000; Wang et al., 2003, Chen et al., 2005), it appears to be limited to ATPases and GTPases. Additional methods are required to probe the function of a wider variety of proteins.
Other investigators have devised alternative strategies to perturb protein function by exploiting existing cellular processes (Banaszynski and Wandless, 2006). For example, Varshavsky and coworkers developed methods for controlling protein function based on the importance of certain N-terminal residues for protein stability (Bachmair et al., 1986). Szostak and coworkers showed that a small peptide sequence could be fused to the N-terminus of a protein of interest to modulate protein stability (Park et al., 1992). Varshavsky and coworkers have further isolated a temperature-sensitive peptide sequence that greatly reduced the half-life or dihydrofolate reductase (DHFR) at the non-permissive temperatures (Dohmen et al., 1994). This approach has been used to study proteins in yeast (Labib et al., 2000; Kanemaki et al., 2003). More recently, several researchers have engineered systems in which dimeric small molecules are used to conditionally target fusion proteins for degradation via E3 ligase or the proteasome, itself (Schneekloth et al., 2004; Janse et al., 2004). However, these systems require either a prior knowledge of the high-affinity ligands that modulate the activity of a protein of interest or they are restricted to genetically engineered yeast strains.
An alternative approach for controlling protein function directly is to interfere with subcellular localization. Several methods are available to regulate protein localization using small-molecule by taking advantage of the FKBP-rapamycin-FRB ternary complex (Kohler and Bertozzi, 2003 and Inoue et al., 2005). Rapamycin and FK506 are potent, commercially available immunosuppressive agents, which are ligands of the FK506-binding protein (FKBP12, FKBP). Rapamycin also binds to FKBP-rapamycin-associated protein (FRAP). FRAP is also called the mammalian target of rapamycin (mTOR), rapamycin and FKBP target 1 (RAFT1), and FRB. Rapamycin binds to and inhibits mTOR by interacting with its FKBP-rapamycin-binding (FRB) domain to inhibit/delay G1 cell cycle progression in mammalian cells (see, e.g., Choi, J. et al. (1996) Science 273:239-42 and Vilella-Bach, M. et al. (1999) J. Biol. Chem. 274:4266-72. The FKBP-rapamycin-binding domain is required for FKBP-rapamycin-associated protein kinase activity and G1 progression. Fusions of proteins of interest can be made to either FKBP or the FRP domain of FRAP/mTOR. Colocalization of the protein of interest is induced upon addition of rapamycin.
Because rapamycin has inherent biological activity, researchers have developed a “bump-hole” strategy (similar to that employed by Shokat and coworkers), wherein rapamycin derivatives possessing large substituents at the FRB binding interface bind poorly to wild-type FRB and in turn the biologically relevant target FRAP/mTOR, with binding restored upon introduction of compensatory cavity-forming mutations in FRB. Specifically, a C20-methallyl-rapamycin derivative (MaRap) binds to a triple-mutated variant of FRB called FRB* (Liberles et al., 1997).
While these methods for regulating protein function directly are noteworthy, there has yet to be described a convenient, general method for regulating protein function, particularly a method that does not require the interaction of multiple proteins. Improved methods for regulating protein function directly, particularly in mammalian cells, are needed.